Genetic sequences encoding substrate-specific dihydroflavonol 4-reductase and uses therefor

ABSTRACT

The invention includes modified dihydroflavonol 4-reductase (DFR) nucleic acids encoding the modified DFR that has altered amino acid sequences at the substrate specificity determining region. The property of the modified DFR is characterized by its ability to reduce dihydrokaempferol (DHK) preferentially over dihydroquercetin (DHQ), and dihydromyricetin (DHM). The invention also includes plants having at least one cell expressing the modified DFR. Such plants are characterized by the increased content of pelargonidin-based pigments. The invention also includes vectors comprising at least a portion of the modified DFR nucleic acids. The invention also includes methods using such vectors for producing plants having the increased content of pelargonidin-based pigments.

BACKGROUND OF THE INVENTION

1. Technical Field of the Invention

The present invention relates to modified DFR nucleic acids and encodingthe modified DFR that preferentially utilize DHK as a substrate andtheir uses for genetically altering plants to increase the content ofpelargonidin-based pigments in the plants.

2. Description of the Prior art

Anthocyanins are classes of pigments that determine flower color andplant pigmentation in angiosperm plants. Among anthocyanins,pelargonidin-based pigments confer bric-red/orange color to plants,while cyanidin- and delphinidin-based pigments confer red and violetcolor each (Holton, et al. Plant Cell 7:1071-1083 (1995); Tanaka, et al.Plant Cell Physiol. 39:1119-1126 (1998)). Different ratio of thesepigments confers a wide range of flower color. Many anthocyaninbiosynthetic genes have been identified. One of key enzyme in thebiosynthetic pathway is dihydroflavonol 4-reductase (DFR). The enzymeconverts dihydroflavonols (dihydrokaempferol (DHK), dihydroquercetin(DHQ), and dihydromyricetin (DHM)) to leucocyanidins. The leucocyanidinsare subsequently converted to anthocyanins by other enzymes. Theconversion of DHK to DHQ and DHM are catalyzed by flavonoid3′-hydroxylase (F3′H) and flavonoid 3′,5′-hydroxylase (F3′5′H). SinceDFRs in most plants can convert all three dihydroflavonols toleucocyanidins, the ratio of three classes of anthocyanin pigments aremainly determined by the activity of F3′H and F3′5′H (Holton, et al.Plant Cell 7:1071-1083 (1995)).

Since pelargonidin-based pigments confer the orange color to flowers,the F3′H and F3′5′H activities must be absent for a plant to have orangecolored flowers (U.S. Pat. No. 5410096). In many plant species, F3′H andF3′5′H are encoded by a multiple genes, thus the mutant lines that lackF3′H and F3′5′H are not easily found. This partially accounts for therarity of orange-colored flowers in some plant species. Inability toreduce DHK to leucocyanidin by DFR in some species can also cause thelack of orange-colored flower. For example, DFRs from Petunia andCymbidium convert DHK to its leucocyanidin very inefficiently, thusthese species do not accumulate large ratio of pelargonidin-basedanthocyanins even if F3′H and F3′5′H are absent (Gerats, et al. Planta155:364-368 (1982); Johnson, et al. Plant J. 19:81-85 (1999)). Anorange-colored Petunia was engineered by introducing a maize DFR to aSpecial mutant line of Petunia that lacks F3′H and F3′5′H (Meyer, et al.Nature 330:677-678 (1987)). Since the maize DFR can convert all threedihydroflavonols to their leucocyanidins, such a mutant line thataccumulates DHK was necessary for the development of orange-coloredPetunia. The necessity of the special mutant line can be circumvented byusing a DFR that utilizes DHK preferentially over DHQ and DHM.

Using chimeric DFRs between Petunia and Gerbera DFRs, we identified aregion that determines the substrate specificity of DFR. By altering anamino acid in the region, we developed a DHK-specific DFR that convertsDHK preferentially over DHQ and DHM. When expressed in plants, theDHK-specific DFR increases the pelargonidin-based pigments regardless ofF3′H activity.

SUMMARY OF THE INVENTION

Accordingly, the object of this invention is to providesubstrate-specific DFRs which have point mutations at residue number 134of SEQ ID NO: 2 when the amino acids are aligned with the ClustalWprogram.

It is an also object herein to provide a DHK-specific DFR and nucleicacids encoding the DHK-specific DFR.

Still further, it is an object herein to provide transgenic plantsexpressing the DHK-specific DFR which confers a phenotype characterizedby the increased content-- of pelargonidin-based pigments in the plants.

In accordance with the objects, the invention includes the modified DFRsand nucleic acids encoding the modified DFRs which have altered aminoacid sequences at the substrate specificity determining region. Theproperties of modified DFRs are characterized by their abilities toreduce one substrate preferentially among DHK, DHQ, and DHM.

The invention also includes a modified DFR that reduces DHKpreferentially over DHQ and DHM.

The invention also includes plants having at least one cell transformedwith a vector comprising at least a portion of the modified DFR nucleicacids. Such plants have a phenotype characterized by the increasedcontent of pelargonidin-based pigments.

The invention also includes vectors capable of transforming a plant cellto increase the content of pelargonidin-based pigments.

The invention also includes methods for producing plants having theincreased content of pelargonidin-based pigments. The methods includessteps of transforming plant cells with vectors containing the modifiedDFR gene; regenerating plants from the transformed cells and selectingthe plant having the increased content of pelargonidin-based-pigments.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic diagram showing three chimeric DFRs. Black barsindicate sequences from a Gerbera DFR and gray bars indicates sequencesfrom a Petunia DFR. Numbers are junctional amino acid positions from thetranslation start site of the Gerbera DFR. C.1, C.2, C.3 are the name ofthree different chimeric DFRs.

FIG. 1B shows representative flowers of transgenic Petunia expressingchimera DFRs or control DFR. Ger indicate the transgenic flowerexpressing Gerbera DFR and C.1, C.2, and C.3 indicate Chimera 1, Chimera2, and Chimera 3 each. RL01 line has a functional Petunia DFR gene. TheC.1 and RL01 bore similar pink colored flowers while others borebric-red colored flower. The transgenic W80 flower expressing C.1 haspink color, while transgenic W80 flowers expressing C.2 and C.3 haveorange/bric-red color. The orange/bric-red color can be also observed inthe transgenic Petunia flowers expressing the native Gerbera DFR.

FIG. 1C shows the TLC analysis data of pigments produced in transgenicPetunia flowers next to standard pigments (pelargonidin (Pg), cyanidin(Cy), and delphinidin (Dp)). The transgenic flowers expressing C.1 hasmainly cyanidin- and delphinidin-based pigments, while the flowersexpressing C.2 and C.3 have mainly pelargonidin-based pigments inaddition to small amount of cyanidin- and delphinidin-based pigments.

FIG. 2 shows the amino acid sequence of Gerbera DFR aligned with otherrepresentative DFR sequences. The ClustalW program was used to alignmultiple amino acid sequences (Thomson, et al. Nucl. Acids Res.22:4673-4680 (1994)). The substrate specificity determining region isboxed and the 134^(th) amino acid residue of Gerbera DFR andcoressponding amino acid residues of DFRs from a few representativespecies are bold typed.

FIG. 3A shows site-directed mutagenesis of substrate specificitydetermining region. The sequence corresponds to the substratespecificity determining region of Gerbera DFR. Arrows and lettersindicates amino acids that were changed to.

FIG. 3B shows flowers of transgenic Petunia expressing mutated GerberaDFR gene. Ger indicates the wild type GerberaDFR and T132V indicates themutated DFR that has valine instead of threonine at the 132^(th)position of Gerbera DFR. Names of other mutated DFRs followed the samenotation rule. All transgenic lines except N134L and E145L have the samebric red colored flower. The N134L bore slightly different coloredflowers and E145L bore white flowers.

FIG. 3C shows a TLC analysis of pigments produced in the transgenicPetunia flowers. As expected, the E145L did not accumulated anyanthocyanin. The N134L accumulated mostly pelargonidin while othermutated DFR and wild type Gebera DFR accumulated significant amount ofcyanidin and delphinidin in addition to perlargonidin.

FIG. 4A shows the development of a DFR that display the alteredsubstrate specificity. WR and WV indicate Petunia lines that aredfr^(−/−), but F3′H^(+/+) (WR) or F3′5′H^(+/+) (WV). The mark −indicatesno DFR gene, DFR^(N134L) indicates DFR that has leucine instead ofaspargine at the 134^(th) position of Gerbera DFR, and DFR^(WT)indicates the wild type Gerbera DFR. The flower located in the crosssection indicate the WR or WV transgenic flowers expressing DFR^(N134L)or DFR^(WT).

FIG. 4B shows a TLC analysis of pigments produced in the transgeniclines. Pg, Cy, and Dp indicate pelargonidin, cyanidin, and delphinidin.The WR and WV lines expressing wild type DFR accumulated cyanidin anddelphinidin each. The WR line expressing DFR^(N134L) accumulatedpelargonidin and cyanidin, while the WV line expressing DFR^(N134L) didnot accumulated any pigment other than background level of delphinidin.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, the substrate specificitydetermining region was identified by determining the abilities of threechimeric DFRs to catalyze the reduction of DHK in the transgenic Petunialines. In order to identify the region of DFR that determines itssubstrate specificity, we constructed chimeric DFR genes using cDNAsequences of Petunia (SEQ ID NO: 35) and Gerbera (SEQ ID NO: 1). Thoughthese two DFRs have high similiarity at the amino acid level, GerberaDFR is able to catalyze dihydrokaempferol (DHK) while Petunia DFR cannot(Elomaa et al. Mol. Gen. Genet. 248:649-656 (1995)). We built threedifferent chimeric genes using regions of high homology as common PCRprimer sites (FIG. 1A). The chimeric genes were transformed into a whiteflowered Petunia mutant (W80) that lacks DFR activity and accumulatesprimarily DHK but with appreciable amounts of dihydroquercetin (DHQ) anddihydromyricetin (DHM) (Huits et al., 1994). Chimera 1 produced pinkflowers while Chimeras 2 and 3 bore orange-pink flowers (FIG. 1B). Thehue of Chimera 1 flowers is very similar to the inbred Petunia mutantRLO1, which has functional DFR activity and accumulates DHK. Thin layerchromatography (TLC) determined that Chimera 1 produced mainly cyanidinand delphinidin (FIG. 1b). Chimeras 2 and 3 primarily producedpelargonidin (FIG. 1C), which is the downstream product of DFR reductionof DHK. These results indicated that the region of DFR conferring theability to reduce DHK was between Chimeras 1 and 2. The identifiedregion (approx. 40 amino acids) is highly variable in DFRs fromdifferent plant species. By excluding the completely conserved aminoacid sequences at the borders, the identified region is narrowed down to26 amino acids. Hereinafter, this region is referred as substratespecificity determining region. An example of the substrate specificitydetermining region in a few representaive DFRs is shown in FIG. 2.

The invention provides the modified DFR nucleic acids and encoded DFRsthat have altered amino acid sequences at the substrate specificitydetermining region. Such DFRs have properties characterized by thealtered substrate specificity. Hereinafter, DFRs that catalyze thereduction of one substrate preferentially over other two substrates arereferred as substrate-specific DFRs. In the preferred embodiments, theinvention provides the modified DFR that has altered amino acid at134^(th) amino acid residue of Gerbera DFR or the corresponding aminoacid residues of DFRs from other species. Such DFRs have propertiescharacterized by catalyzing the reduction of DHK preferentially over DHQand DHM. Hereinafter, DFRs that catalyze the reduction of DHKpreferentially over DHQ and DHM are referred as DHK-specific DFRs. The134^(th) amino acid residue of Gerbera DFR and corresponding amino acidresidues of DFRs from a few representative species are shown in FIG. 2.

In accordance with the present invention, a DHK-specific DFR wasdeveloped by replacing asparagine at 134^(th) amino acid residue ofGerbera DFR to leucine. The expression of the DHK-specific DFR in W80Petunia line, which accumulates large amount of DHK in addition toappreciable amount of DHQ and DHM, caused the production of onlypelargonidin. The expression of native Gerbera DFR in the same Petunialine caused the production of appreciable amounts of cyanidin anddelphinidin in addition to pelargonidin (FIG. 3). Since the W80 Petunialine we transformed accumulates mainly DHK with small amount of DHQ andDHM, it was not clear if the N134L mutant DFR completely lost thecapability of reducing DHQ and DHM. To investigate if the N134L mutantDFR produces only pelargonidin in the presence of fully activeflavonoid-3′-hydroxylase (F3′H) or flavonoid-3′, 5′-hydroxylase(F3′5′H), we crossed our N134L transformant with Petunia lines that areeither dfr^(−/−)/F3′H^(+/+) (WR line) or dfr^(−/−)/F3′5′H^(+/+) (WVline). As shown in FIG. 4A, both WR and WV lines bore white flowers asexpected. When these lines were crossed with the N134L transformants,the WR line expressing the mutant DFR (WR/DFR^(N134L)) had orangecolored flowers while the WR expressing wild type Gerbera DFR(WR/DFR^(WT)) had red colored flowers. Unlike the WR lines, the WV linesexpressing the mutant DFR (WV/DFR^(N134L)) bore white flowers while WVlines expressing the wild type DFR (WV/DFR^(WT)) had violet coloredflowers. To determine the pigments produced in these crossed lines, weperformed TLC analysis. FIG. 4B shows that the WR/DFR^(N134L)accumulated a large amount of pelargonidin while WR/DFR^(WT) mainlyaccumulated cyanidin. In the white flowered WV/DFR^(N134L), noappreciable amounts of anthocyanidins accumulated other than abackground level of delphinidin. In contrast to WV/DFR^(N134L), theWV/DFR^(WT) accumulated mainly delphinidin. The data indicate that theN134L mutant DFR preferentially utilizes DHK as a substrate over DHQ andcannot reduce DHM. The substrate preference of the N134L mutant DFR issomewhat opposite to that of Petunia DFR which prefer DHM over DHQ andcannot use DHK (Forkmann and Ruhnau, 1987).The results indicates thatthe DHK-specific DFR can increase the pelargonidin-based pigments inplants regardless of the presence of F3′H activity.

The invention also provides plants having cells transformed with vectorscomprising at least a portion of the substrate-specific DFR nucleicacids. Such plants have phenotypes characterized by the increasedcontent of anthocyanins specified by the substrate specific DFRs. In thepreferred embodiments, the invention provides plants having cellstransformed with vectors comprising at least a portion of theDHK-specific DFR nucleic acids. Such plants have phenotypescharacterized by the increased content of pelargonidin-based pigments.Plants that can be used to practice the invention include plants withinthe Division of Magnoliphyta, i.e. the angiosperms include thedicotyledons and the monocotyledons. Particularly preferred Orders ofangiosperms according to “Plant Systematics”, S. B. Jones, Jr. and A. E.Luchsinger include Magnoliales, Laurales, Aristolochiales, Nymphaeales,Ranunculales, Caryophyllales, Malvales, Violales, Capparales, Ericales,Primulales, Rosales, Fabales, Myrtales, Cornales, Rhamnales, Sapindales,Geraniales, Apiales, Gentianales, Solanales, Lamiales, Scrophulariales,Campanulales, Rubiales, Dipsacales, Asterales, Hydrocharitales, Arales,Cyperales, Liliales, and Orchidales. Particularly preferred plantsinclude orchid, iris, campanula, gentiana, phlox, cyclamen, eustoma,crocus, delphinium, ageratum, chrysanthemum, Petunia, cactus, limonium,astilbe, carnation, Gerbera, brassica, impatience, geranium, dahlia,sunflower, dianthus, gloxinia, caledula, bellis, ranunculus, aster,tagetes, salvia, hibiscus, cirsium, godetia, catharanthus, alyssum,lupinus, portulaca, drotheanthus, tulip, lily, narcissus, freesia,anemone, gladiolus, caladium, archimenes, achillea, agapanthus,aethiones, allium, alstroemeria, amaryllis, anagallis, androsace,anemone, antirrhinum, aquilegia, armeria, asperula, begonia, browallia,callistephus, camellia, ceanothus, chionodoxa, cistus, clarkia,clematis, colchicun, consolida, cornus, cosmos, deutzia, digitalis,erigeron, erodium, erysimum, erythronium, felicia, gazania, gypsophila,helenium, helianthemum, heliophila, hippeastrum, hyacinthus, hydrangea,iberis, ipomoea, ixia, jacaranda, kalmia, kolkwitzia, lagerstroemia,lathyrus, lavatera, legousia, lewsia, linum, lobelia, lobularia,magnolia, malus, malva, mathiola, merendera, mimulus, myosotis,narcissus, nemesia, nicotiana, nopalxochia, nymphaea, omphalodes,orthrosanthus, osteospermum, oxalis, paeonia, pelargonium, penstemon,pentas, pericallis, persicaria, platycodon, polemonium, polygala,potentilla, primula, prunus, puschkinia, rhododendron, rhodohypoxis,rose, saintpaulia, saponaria, saxifraga, scabiosa, schizostylis,schlumbergera, schilla, sedum, senecio, silene, solanum, spiraea,stachys, streptocarpus, syringa, tagetes, tanacetum, thunbergia, thymus,torenia, tropaeolum, verbena, veronica, viburnum, vinca, viola, vitis,watsonia, and zinnia. The broad applicability of the modified DFRnucleic acids is based on the universal function of DFR in anthocyaninbiosynthesis in divergent plant taxa. The parent plant used to practicethe invention can be a wild type variant, a mutant which has beengenerated by the mutagenesis, or a transgenic line that has beengenerated by the recombinant techniques.

The invention also provides plant transformation vectors comprising atleast a portion of substrate-specific DFR nucleic acids. In thepreferred embodiments, the invention provides a plant transformationvector comprising at least a portion of DHK-specific DFR nucleic acids.Particularly preferred promoter to drive the expression of theDHK-specific DFR nucleic acids is the cauliflower mosaic virus 35Sprotein promoter. However, other constitutive promoters, tissue specificpromoters, or inducible promoters can be also used.

The transformation of plants can be carried out in accordance with theinvention by any of various transformation methods known to thoseskilled in the art of plant molecular biology. Particular methods fortransformation include the transfer of nucleic acids into a plant cellby the microinjection, polyethylene glycol, electroporation, ormicrobombardment. Alternatively, plant cells can be transformed byAgrobacterium harboring vectors comprising at least a portion ofmodified DFR nucleic acids.

Regeneration of plants from the transformed cells can be carried out byany methods known to those skilled in the art. See, e.g., Methods inEnzymology, supra.; Methods in Enzymology, Vol 118; and Klee et al.Annual Review of Plant Physiology 38:467-486. Transformed cells orplants are selected based on their resistance to certain chemicals suchas antibiotics or based on their phenotypes characterized by theincreased content of pelargonidin-based pigments. The transformed plantscan be self-fertilized or crossed with other plants. After thefertilization, the plants expressing at least portion of the modifiedDFR nucleic acids can be selected based on their resistance to certainchemicals such as antibiotics or based on their phenotypes characterizedby the increased content of pelargonidin-based pigments. Alternatively,the transformed cells or a part of transformed plants can be grafted toother plants.

The following is presented as examples and is not to be construed as alimitation on the scope of the invention.

EXAMPLE Petunia Transformation

Leaf explants of the inbred Petunia W80 line (an6⁻, ht1⁻, ht2⁻, hf1⁻,hf2⁻, f1⁻, and rf⁻) were transformed as described elsewhere except thatleaf explants recently infected by Agrobacterium tumefaciens were rinsedwith Murashige-Skoog solution containing 750 mg/L cefotaxime and thenplaced on media having 100 mg/L kanamycin sulfate and 500 mg/Lcefotaxime(Johnson, et al. Plant J. 19:81-85 (1999)). Also, putativetransformants were grown on MS media with vitamins, 30 g/L sucrose, 0.6%agar and 500 mg/L cefotaxime; after rooting the transformants weretransferred to soil.

Chimeric Gene Construction

Highly conserved regions of the DFR gene were identified by a multiplesequence alignment of a number of DFRs. The 5′ region (Gerbera DFRportion) of each chimeric gene was synthesized from the Gerbera DFR cDNAclone using a primer containing the codon for the starting methionine ofthe Gerbera DFR gene (SEQ ID NO. 5): 5′-GGC GAA AAT GGA AGA GGA TTCTCC-3′ and a primer containing a conserved region of the Gerbera DFRgene (Chimera 1; SEQ ID NO: 6: 5′-AGC AGA TGA AGT GAA CAC TAG TTT CTTCAC-3′; Chimera 2; SEQ ID NO: 7: 5′-GGC TTT CTC TGC CAG AGT TTT TGA CACGAA-3′; Chimera 3; SEQ ID NO: 8: 5′-GTG GGA CGA GCA AAT GTA TCT TCC TTTTGC-3 ′). The 3′ region (Petunia DFR portion) of each chimeric gene wassynthesized from the Petunia DFRA cDNA clone using a primercomplementary to the three conserved regions (Chimera 1; SEQ ID NO: 9:5′- TTC ACT TCA TCT GCT GGA ACT CTC GAT GTG; Chimera 2; SEQ ID NO: 10:5′-CTG GCA GAG AAA GCC GCA ATG GAA GAA GCT-3′; Chimera 3; SEQ ID NO: 11:5′-ATT TGC TCG TCC CAC CAT GCT ATC ATC TAC-3′) and a primer containingthe stop codon of the Petunia DFRA gene (SEQ ID NO: 12): 5′-GCG CTA GACTTC AAC ATT GCT T AA-3 ′5′ and 3′ regions were gel purified after PCRamplification. To assemble the full length chimeric gene the 5′ and 3′region fragments were added to the same tube in roughly equal amountsand subjected to PCR cycles (94° C. 30″, 55° C., 30″, 72° C. 1:30).Full-length chimeric genes (−1.1 kb ) were purified from agarose gels.The chimeric genes were cloned into a vector containing the 35S CaMVpromoter and NOS terminator. Pfu polymerase (Stratagene, La Jolla,Calif.) was used for all PCR reactions.

Amino Acid Point Mutant Construction

Gerbera DFR genes containing one amino acid point mutation were made ina similar manner as the chimeric genes. The 5′ region was synthesizedusing a primer having the Gerbera DFR starting methionine and a primercontaining a single codon change. The 3′ region was made with acomplementary primer with the single codon change and a primer havingthe stop codon of Gerbera DFR. The full length mutant sequence wasassembled like the chimeric genes above. Each point mutant was clonedinto a vector having the 35S CaMV promoter and NOS terminator. Themutagenized region of each mutant DFR was sequenced to ensure thecorrect residue was changed. Point mutants were then transformed intothe W80 Petunia line. The transformants expressing the DFR genes werecrossed with WR Petunia line (dfr^(−/−), F3′H^(+/+)) and WV Petunia line(dfr^(−/−), F3′5′H^(+/+)) to determine the substrate specificity of themutated DFR. Mutations in other loci were not determined in these twoPetunia lines.

TLC Analysis

Anthocyanidins were separated on cellulose TLC plates as described(Johnson, et al. Plant J. 19:81-85 (1999)). Corollas were sometimesstored at 4° C. for extended periods of time in methanol-0.5% HClsolution. Before adding iso-amylalcohol, the flower extracts werequantified at 530 nm to ensure uniform loading on the TLC plate.Anthocyanin standards were purchased from Apin Chemicals Ltd.(Oxfordshire, England).

Sequence Alignment

Multiple sequence alignment of DFRs was done using ClustalW program.

SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 35 <210> SEQ ID NO 1 <211>LENGTH: 1101 <212> TYPE: DNA <213> ORGANISM: Gerbera sp. <300>PUBLICATION INFORMATION: <301> AUTHORS: Helariutta, Y., Kotilainen, M.,Elomaa, P. and Teeri, T. H. <302> TITLE: Gerbera hybrida (Asteraceae)imposes regulation at several anatomical levels during inflorescencedevelopment on the gene for dihydroflavanol-4-reductase <303> JOURNAL:Plant Mol. Biol. 28(5), 935-41 <304> VOLUME: 28 <305> ISSUE: 5 <306>PAGES: 935-941 <307> DATE: 1995-__-__ <308> DATABASE ACCESSION NUMBER:Z17221 <309> DATABASE ENTRY DATE: 1995-11-23 <313> RELEVANT RESIDUES:(31)..(1131) <400> SEQUENCE: 1 atggaagagg attctccggc caccgtttgtgtcaccggag cggccgggtt catcggctca 60 tggctcgtca tgagacttct tgaacgtggatacgttgttc atgcaactgt tcgtgatccc 120 ggtgacttga agaaggtgaa gcatttgctagaactaccaa aagcacaaac aaacttgaaa 180 ttatggaaag cagatttgac acaagaaggaagctttgatg aagccattca aggttgccat 240 ggtgtcttcc atctggccac tcctatggactttgagtcca aggaccctga gaacgaaatt 300 ataaagccaa caatcgaagg ggtattaagcatcattcgat catgtgtcaa agcgaaaacc 360 gtgaagaaac tagtgttcac ctcctccgccgggaccgtga acggacaaga gaaacaactg 420 cacgtgtacg acgaatctca ttggagcgatttggatttta tatactctaa aaaaatgact 480 gcttggatgt atttcgtgtc aaaaactttggctgaaaaag ctgcgtggga tgcaacgaaa 540 ggaaacaaca ttagttttat tagtatcatcccaaccctgg tagttggtcc gtttatcacc 600 tcgacgttcc caccaagtct cgttaccgcgctttctttga tcacgggcaa tgaagcacat 660 tattcaatta taaagcaagg tcaatatgtgcacttagatg atctttgtga gtgtcatata 720 tacctatatg agaaccctaa agcaaaaggaagatacattt gttcttctca tgatgccacc 780 attcatcaat tggctaaaat catcaaagacaagtggccag agtactatat tccaaccaag 840 tttccgggga tcgatgagga gctaccgatagtttcttttt cgtcaaagaa gttaattgac 900 acgggtttcg agtttaagta taatttagaggacatgttta aaggagccat tgatacatgt 960 agagaaaagg gattgcttcc atattccacaatcaagaacc atataaatgg taaccatgtt 1020 aatggtgttc atcattatat aaaaaacaatgatgatgatc atgaaaaggg tttgctttgt 1080 tgttcaaaag aaggccaata g 1101 <210>SEQ ID NO 2 <211> LENGTH: 366 <212> TYPE: PRT <213> ORGANISM: Gerberasp. <400> SEQUENCE: 2 Met Glu Glu Asp Ser Pro Ala Thr Val Cys Val ThrGly Ala Ala Gly 1 5 10 15 Phe Ile Gly Ser Trp Leu Val Met Arg Leu LeuGlu Arg Gly Tyr Val 20 25 30 Val His Ala Thr Val Arg Asp Pro Gly Asp LeuLys Lys Val Lys His 35 40 45 Leu Leu Glu Leu Pro Lys Ala Gln Thr Asn LeuLys Leu Trp Lys Ala 50 55 60 Asp Leu Thr Gln Glu Gly Ser Phe Asp Glu AlaIle Gln Gly Cys His 65 70 75 80 Gly Val Phe His Leu Ala Thr Pro Met AspPhe Glu Ser Lys Asp Pro 85 90 95 Glu Asn Glu Ile Ile Lys Pro Thr Ile GluGly Val Leu Ser Ile Ile 100 105 110 Arg Ser Cys Val Lys Ala Lys Thr ValLys Lys Leu Val Phe Thr Ser 115 120 125 Ser Ala Gly Thr Val Asn Gly GlnGlu Lys Gln Leu His Val Tyr Asp 130 135 140 Glu Ser His Trp Ser Asp LeuAsp Phe Ile Tyr Ser Lys Lys Met Thr 145 150 155 160 Ala Trp Met Tyr PheVal Ser Lys Thr Leu Ala Glu Lys Ala Ala Trp 165 170 175 Asp Ala Thr LysGly Asn Asn Ile Ser Phe Ile Ser Ile Ile Pro Thr 180 185 190 Leu Val ValGly Pro Phe Ile Thr Ser Thr Phe Pro Pro Ser Leu Val 195 200 205 Thr AlaLeu Ser Leu Ile Thr Gly Asn Glu Ala His Tyr Ser Ile Ile 210 215 220 LysGln Gly Gln Tyr Val His Leu Asp Asp Leu Cys Glu Cys His Ile 225 230 235240 Tyr Leu Tyr Glu Asn Pro Lys Ala Lys Gly Arg Tyr Ile Cys Ser Ser 245250 255 His Asp Ala Thr Ile His Gln Leu Ala Lys Ile Ile Lys Asp Lys Trp260 265 270 Pro Glu Tyr Tyr Ile Pro Thr Lys Phe Pro Gly Ile Asp Glu GluLeu 275 280 285 Pro Ile Val Ser Phe Ser Ser Lys Lys Leu Ile Asp Thr GlyPhe Glu 290 295 300 Phe Lys Tyr Asn Leu Glu Asp Met Phe Lys Gly Ala IleAsp Thr Cys 305 310 315 320 Arg Glu Lys Gly Leu Leu Pro Tyr Ser Thr IleLys Asn His Ile Asn 325 330 335 Gly Asn His Val Asn Gly Val His His TyrIle Lys Asn Asn Asp Asp 340 345 350 Asp His Glu Lys Gly Leu Leu Cys CysSer Lys Glu Gly Gln 355 360 365 <210> SEQ ID NO 3 <211> LENGTH: 1101<212> TYPE: DNA <213> ORGANISM: Gerbera sp. <400> SEQUENCE: 3 atggaagaggattctccggc caccgtttgt gtcaccggag cggccgggtt catcggctca 60 tggctcgtcatgagacttct tgaacgtgga tacgttgttc atgcaactgt tcgtgatccc 120 ggtgacttgaagaaggtgaa gcatttgcta gaactaccaa aagcacaaac aaacttgaaa 180 ttatggaaagcagatttgac acaagaagga agctttgatg aagccattca aggttgccat 240 ggtgtcttccatctggccac tcctatggac tttgagtcca aggaccctga gaacgaaatt 300 ataaagccaacaatcgaagg ggtattaagc atcattcgat catgtgtcaa agcgaaaacc 360 gtgaagaaactagtgttcac ctcctccgcc gggaccgtgc tcggacaaga gaaacaactg 420 cacgtgtacgacgaatctca ttggagcgat ttggatttta tatactctaa aaaaatgact 480 gcttggatgtatttcgtgtc aaaaactttg gctgaaaaag ctgcgtggga tgcaacgaaa 540 ggaaacaacattagttttat tagtatcatc ccaaccctgg tagttggtcc gtttatcacc 600 tcgacgttcccaccaagtct cgttaccgcg ctttctttga tcacgggcaa tgaagcacat 660 tattcaattataaagcaagg tcaatatgtg cacttagatg atctttgtga gtgtcatata 720 tacctatatgagaaccctaa agcaaaagga agatacattt gttcttctca tgatgccacc 780 attcatcaattggctaaaat catcaaagac aagtggccag agtactatat tccaaccaag 840 tttccggggatcgatgagga gctaccgata gtttcttttt cgtcaaagaa gttaattgac 900 acgggtttcgagtttaagta taatttagag gacatgttta aaggagccat tgatacatgt 960 agagaaaagggattgcttcc atattccaca atcaagaacc atataaatgg taaccatgtt 1020 aatggtgttcatcattatat aaaaaacaat gatgatgatc atgaaaaggg tttgctttgt 1080 tgttcaaaagaaggccaata g 1101 <210> SEQ ID NO 4 <211> LENGTH: 366 <212> TYPE: PRT<213> ORGANISM: Gerbera sp. <300> PUBLICATION INFORMATION: <308>DATABASE ACCESSION NUMBER: Z17221 <309> DATABASE ENTRY DATE: 1995-11-23<400> SEQUENCE: 4 Met Glu Glu Asp Ser Pro Ala Thr Val Cys Val Thr GlyAla Ala Gly 1 5 10 15 Phe Ile Gly Ser Trp Leu Val Met Arg Leu Leu GluArg Gly Tyr Val 20 25 30 Val His Ala Thr Val Arg Asp Pro Gly Asp Leu LysLys Val Lys His 35 40 45 Leu Leu Glu Leu Pro Lys Ala Gln Thr Asn Leu LysLeu Trp Lys Ala 50 55 60 Asp Leu Thr Gln Glu Gly Ser Phe Asp Glu Ala IleGln Gly Cys His 65 70 75 80 Gly Val Phe His Leu Ala Thr Pro Met Asp PheGlu Ser Lys Asp Pro 85 90 95 Glu Asn Glu Ile Ile Lys Pro Thr Ile Glu GlyVal Leu Ser Ile Ile 100 105 110 Arg Ser Cys Val Lys Ala Lys Thr Val LysLys Leu Val Phe Thr Ser 115 120 125 Ser Ala Gly Thr Val Leu Gly Gln GluLys Gln Leu His Val Tyr Asp 130 135 140 Glu Ser His Trp Ser Asp Leu AspPhe Ile Tyr Ser Lys Lys Met Thr 145 150 155 160 Ala Trp Met Tyr Phe ValSer Lys Thr Leu Ala Glu Lys Ala Ala Trp 165 170 175 Asp Ala Thr Lys GlyAsn Asn Ile Ser Phe Ile Ser Ile Ile Pro Thr 180 185 190 Leu Val Val GlyPro Phe Ile Thr Ser Thr Phe Pro Pro Ser Leu Val 195 200 205 Thr Ala LeuSer Leu Ile Thr Gly Asn Glu Ala His Tyr Ser Ile Ile 210 215 220 Lys GlnGly Gln Tyr Val His Leu Asp Asp Leu Cys Glu Cys His Ile 225 230 235 240Tyr Leu Tyr Glu Asn Pro Lys Ala Lys Gly Arg Tyr Ile Cys Ser Ser 245 250255 His Asp Ala Thr Ile His Gln Leu Ala Lys Ile Ile Lys Asp Lys Trp 260265 270 Pro Glu Tyr Tyr Ile Pro Thr Lys Phe Pro Gly Ile Asp Glu Glu Leu275 280 285 Pro Ile Val Ser Phe Ser Ser Lys Lys Leu Ile Asp Thr Gly PheGlu 290 295 300 Phe Lys Tyr Asn Leu Glu Asp Met Phe Lys Gly Ala Ile AspThr Cys 305 310 315 320 Arg Glu Lys Gly Leu Leu Pro Tyr Ser Thr Ile LysAsn His Ile Asn 325 330 335 Gly Asn His Val Asn Gly Val His His Tyr IleLys Asn Asn Asp Asp 340 345 350 Asp His Glu Lys Gly Leu Leu Cys Cys SerLys Glu Gly Gln 355 360 365 <210> SEQ ID NO 5 <211> LENGTH: 24 <212>TYPE: DNA <213> ORGANISM: Gerbera sp. <400> SEQUENCE: 5 ggcgaaaatggaagaggatt ctcc 24 <210> SEQ ID NO 6 <211> LENGTH: 30 <212> TYPE: DNA<213> ORGANISM: Gerbera sp. <400> SEQUENCE: 6 agcagatgaa gtgaacactagtttcttcac 30 <210> SEQ ID NO 7 <211> LENGTH: 31 <212> TYPE: DNA <213>ORGANISM: Gerbera sp. <400> SEQUENCE: 7 ggctttctct gccagagttt ttgagcacgaa 31 <210> SEQ ID NO 8 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM:Gerbera sp. <400> SEQUENCE: 8 gtgggacgag caaatgtatc ttccttttgc 30 <210>SEQ ID NO 9 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Petunia sp.<400> SEQUENCE: 9 ttcacttcat ctgctggaac tctcgatgtg 30 <210> SEQ ID NO 10<211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Petunia sp. <400>SEQUENCE: 10 ctggcagaga aagccgcaat ggaagaagct 30 <210> SEQ ID NO 11<211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Petunia sp. <400>SEQUENCE: 11 atttgctcgt cccaccatgc tatcatctac 30 <210> SEQ ID NO 12<211> LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Petunia sp. <400>SEQUENCE: 12 gcgctagact tcaacattgc ttaa 24 <210> SEQ ID NO 13 <211>LENGTH: 60 <212> TYPE: PRT <213> ORGANISM: Lilium sp. <400> SEQUENCE: 13Lys Ala Gly Thr Val Lys Arg Val Ile Phe Thr Ser Ser Ala Gly Thr 1 5 1015 Val Asn Val Gln Glu Asn Gln Met Pro Glu Tyr Asp Glu Ser Ser Trp 20 2530 Ser Asp Val Asp Phe Cys Arg Arg Val Lys Met Thr Gly Trp Met Tyr 35 4045 Phe Val Ser Lys Thr Leu Ala Glu Lys Ala Ala Trp 50 55 60 <210> SEQ IDNO 14 <211> LENGTH: 60 <212> TYPE: PRT <213> ORGANISM: Hordeum sp. <400>SEQUENCE: 14 Glu Ala Gly Thr Val Lys Arg Ile Val Phe Thr Ser Ser Ala GlySer 1 5 10 15 Val Asn Ile Glu Glu Arg Pro Arg Pro Ala Tyr Asp Gln AspAsn Trp 20 25 30 Ser Asp Ile Asp Tyr Cys Arg Arg Val Lys Met Thr Gly TrpMet Tyr 35 40 45 Phe Val Ser Lys Ala Leu Ala Glu Lys Ala Ala Met 50 5560 <210> SEQ ID NO 15 <211> LENGTH: 60 <212> TYPE: PRT <213> ORGANISM:Antirrhinum sp. <400> SEQUENCE: 15 Gln Ala Lys Thr Val Lys Lys Phe IlePhe Thr Thr Ser Gly Gly Thr 1 5 10 15 Val Asn Val Glu Glu His Gln LysPro Val Tyr Asp Glu Thr Asp Ser 20 25 30 Ser Asp Met Asp Phe Ile Asn SerLys Lys Met Thr Gly Trp Met Tyr 35 40 45 Phe Val Ser Lys Ile Leu Ala GluLys Ala Gly Met 50 55 60 <210> SEQ ID NO 16 <211> LENGTH: 60 <212> TYPE:PRT <213> ORGANISM: Petunia sp. <400> SEQUENCE: 16 Lys Ala Asn Thr ValLys Arg Leu Val Phe Thr Ser Ser Ala Gly Thr 1 5 10 15 Leu Asp Val GlnGlu Gln Gln Lys Leu Phe Tyr Asp Gln Thr Ser Trp 20 25 30 Ser Asp Leu AspPhe Ile Tyr Ala Lys Lys Met Thr Gly Trp Met Tyr 35 40 45 Phe Ala Ser LysIle Leu Ala Glu Lys Ala Ala Met 50 55 60 <210> SEQ ID NO 17 <211>LENGTH: 60 <212> TYPE: PRT <213> ORGANISM: Callistephus sp. <400>SEQUENCE: 17 Lys Ala Lys Thr Val Lys Lys Leu Val Tyr Thr Ser Ser Ala GlyThr 1 5 10 15 Val Asn Val Gln Glu Thr Gln Leu Pro Val Tyr Asp Glu SerHis Trp 20 25 30 Ser Asp Leu Asp Phe Ile Tyr Ser Lys Lys Met Thr Ala TrpMet Tyr 35 40 45 Phe Val Ser Lys Thr Leu Ala Glu Lys Ala Ala Met 50 5560 <210> SEQ ID NO 18 <211> LENGTH: 60 <212> TYPE: PRT <213> ORGANISM:Dacus sp. <400> SEQUENCE: 18 Lys Ala Lys Thr Val Lys Lys Leu Ile Tyr ThrSer Ser Ala Gly Thr 1 5 10 15 Val Asn Val Arg Glu His Gln Leu Pro ValTyr Asp Glu Ser Asn Trp 20 25 30 Ser Asp Met Asp Phe Ile Tyr Ser Thr LysMet Thr Ala Trp Met Tyr 35 40 45 Phe Val Ser Lys Ser Leu Ala Glu Lys AlaAla Trp 50 55 60 <210> SEQ ID NO 19 <211> LENGTH: 60 <212> TYPE: PRT<213> ORGANISM: Camellia sp. <400> SEQUENCE: 19 Lys Ala Lys Thr Val LysArg Leu Val Phe Thr Ser Ser Ala Gly Thr 1 5 10 15 Val Asn Val Gln GluHis Gln Gln Pro Val Phe Asp Glu Asn Asn Trp 20 25 30 Ser Asp Leu Asp PheIle Asn Lys Lys Lys Met Thr Gly Trp Met Tyr 35 40 45 Phe Val Ser Lys ThrLeu Ala Glu Lys Ala Ala Trp 50 55 60 <210> SEQ ID NO 20 <211> LENGTH: 60<212> TYPE: PRT <213> ORGANISM: Arabidopsis sp. <400> SEQUENCE: 20 LysAla Lys Thr Val Arg Arg Phe Val Phe Thr Ser Ser Ala Gly Thr 1 5 10 15Val Asn Val Glu Glu His Gln Lys Asn Val Tyr Asp Glu Asn Asp Trp 20 25 30Ser Asp Leu Glu Phe Ile Met Ser Lys Lys Met Thr Gly Trp Met Tyr 35 40 45Phe Val Ser Lys Ser Leu Ala Glu Lys Ala Ala Trp 50 55 60 <210> SEQ ID NO21 <211> LENGTH: 60 <212> TYPE: PRT <213> ORGANISM: Gentiana sp. <400>SEQUENCE: 21 Lys Ala Lys Thr Val Lys Lys Leu Val Phe Thr Ser Ser Ala GlyThr 1 5 10 15 Val Asp Val Gln Glu Gln Gln Lys Pro Val Tyr Asp Glu AsnAsp Trp 20 25 30 Ser Asp Leu Asp Phe Ile Asn Ser Thr Lys Met Thr Gly TrpMet Tyr 35 40 45 Phe Val Ser Lys Ile Leu Ala Glu Lys Ala Ala Trp 50 5560 <210> SEQ ID NO 22 <211> LENGTH: 60 <212> TYPE: PRT <213> ORGANISM:Ipomoea sp. <400> SEQUENCE: 22 Lys Ala Lys Thr Val Lys Arg Leu Val PheThr Ser Ser Ala Gly Thr 1 5 10 15 Leu Asn Val Gln Pro Gln Gln Lys ProVal Tyr Asp Glu Ser Cys Trp 20 25 30 Ser Asp Leu Asp Phe Ile Tyr Ala LysLys Met Thr Gly Trp Met Tyr 35 40 45 Phe Ala Ser Lys Ile Leu Ala Glu LysGlu Ala Trp 50 55 60 <210> SEQ ID NO 23 <211> LENGTH: 59 <212> TYPE: PRT<213> ORGANISM: Vitis sp. <400> SEQUENCE: 23 Ala Lys Thr Val Arg Arg LeuVal Phe Thr Ser Ser Ala Gly Thr Val 1 5 10 15 Asn Ile Gln Glu His GlnLeu Pro Val Tyr Asp Glu Ser Cys Trp Ser 20 25 30 Asp Met Glu Phe Cys ArgAla Lys Lys Met Thr Ala Trp Met Tyr Phe 35 40 45 Val Ser Lys Thr Leu AlaGlu Gln Ala Ala Trp 50 55 <210> SEQ ID NO 24 <211> LENGTH: 60 <212>TYPE: PRT <213> ORGANISM: Forsythia sp. <400> SEQUENCE: 24 Lys Ala LysThr Val Lys Arg Ile Val Phe Thr Ser Ser Ala Gly Thr 1 5 10 15 Val AsnVal Glu Glu His Gln Lys Ser Val Tyr Asp Glu Thr Asp Tyr 20 25 30 Ser AspLeu Asn Phe Ile Tyr Ser Lys Lys Met Thr Gly Trp Met Tyr 35 40 45 Phe ValSer Lys Ile Leu Ala Glu Lys Val Ala Trp 50 55 60 <210> SEQ ID NO 25<211> LENGTH: 60 <212> TYPE: PRT <213> ORGANISM: Lycopersicon sp. <400>SEQUENCE: 25 Lys Ala Asn Thr Val Lys Arg Leu Val Phe Thr Ser Ser Ala GlyThr 1 5 10 15 Leu Asp Val Gln Glu Asp Gln Lys Leu Phe Tyr Asp Glu ThrSer Trp 20 25 30 Ser Asp Leu Asp Phe Ile Tyr Ala Lys Lys Met Thr Gly TrpMet Tyr 35 40 45 Phe Val Ser Lys Ile Leu Ala Glu Lys Ala Ala Met 50 5560 <210> SEQ ID NO 26 <211> LENGTH: 60 <212> TYPE: PRT <213> ORGANISM:Bromheadia sp. <400> SEQUENCE: 26 Lys Ala Gly Ser Val Lys Arg Val IlePhe Thr Ser Ser Ala Gly Thr 1 5 10 15 Val Asn Val Glu Glu His Gln AlaAla Val Tyr Asp Glu Asn Ser Trp 20 25 30 Ser Asp Leu His Phe Val Thr ArgVal Lys Met Thr Gly Trp Met Tyr 35 40 45 Phe Val Ser Lys Thr Leu Ala GluLys Ala Ala Trp 50 55 60 <210> SEQ ID NO 27 <211> LENGTH: 60 <212> TYPE:PRT <213> ORGANISM: Lotus sp. <400> SEQUENCE: 27 Lys Ala Lys Thr Val GlnArg Leu Val Phe Thr Ser Ser Ala Gly Thr 1 5 10 15 Leu Asn Ala Val GluHis Gln Lys Gln Met Tyr Asp Glu Ser Cys Trp 20 25 30 Ser Asp Val Glu PheCys Arg Arg Val Lys Met Thr Gly Trp Met Tyr 35 40 45 Phe Val Ser Lys ThrLeu Ala Glu Gln Glu Ala Trp 50 55 60 <210> SEQ ID NO 28 <211> LENGTH: 60<212> TYPE: PRT <213> ORGANISM: Rosa sp. <400> SEQUENCE: 28 Lys Ala LysThr Val Arg Arg Leu Val Phe Thr Ser Ser Ala Gly Ser 1 5 10 15 Val AsnVal Glu Glu Thr Gln Lys Pro Val Tyr Asn Glu Ser Asn Trp 20 25 30 Ser AspVal Glu Phe Cys Arg Arg Val Lys Met Thr Gly Trp Met Tyr 35 40 45 Phe AlaSer Lys Thr Leu Ala Glu Gln Glu Ala Trp 50 55 60 <210> SEQ ID NO 29<211> LENGTH: 60 <212> TYPE: PRT <213> ORGANISM: Glycine sp. <400>SEQUENCE: 29 Lys Ala Lys Thr Val Arg Arg Leu Ile Phe Thr Ser Ser Ala GlyThr 1 5 10 15 Leu Asn Val Ile Glu Arg Gln Lys Pro Val Phe Asp Asp ThrCys Trp 20 25 30 Ser Asp Val Glu Phe Cys Arg Arg Val Lys Met Thr Gly TrpMet Tyr 35 40 45 Phe Val Ser Lys Thr Leu Ala Glu Lys Glu Ala Trp 50 5560 <210> SEQ ID NO 30 <211> LENGTH: 59 <212> TYPE: PRT <213> ORGANISM:Zea sp. <400> SEQUENCE: 30 Glu Ala Gly Thr Val Arg Arg Ile Val Phe ThrSer Ser Ala Gly Thr 1 5 10 15 Val Asn Leu Glu Glu Arg Gln Arg Pro ValTyr Asp Glu Glu Ser Trp 20 25 30 Thr Asp Val Asp Phe Cys Arg Arg Val LysMet Thr Gly Trp Met Tyr 35 40 45 Phe Val Ser Lys Thr Leu Ala Glu Lys AlaAla 50 55 <210> SEQ ID NO 31 <211> LENGTH: 59 <212> TYPE: PRT <213>ORGANISM: Sorghum sp. <400> SEQUENCE: 31 Glu Ala Gly Thr Val Arg Arg IleVal Phe Thr Ser Ser Ala Gly Thr 1 5 10 15 Val Asn Ile Glu Glu Arg GlnArg Pro Val Tyr Asp Gln Asp Asn Trp 20 25 30 Ser Asp Val Asp Phe Cys GlnArg Val Lys Met Thr Gly Trp Met Tyr 35 40 45 Phe Val Ser Lys Ser Leu AlaGlu Lys Ala Ala 50 55 <210> SEQ ID NO 32 <211> LENGTH: 60 <212> TYPE:PRT <213> ORGANISM: Medicago sp. <400> SEQUENCE: 32 Lys Ala Lys Thr ValArg Arg Leu Ile Tyr Thr Ser Ser Ala Gly Thr 1 5 10 15 Leu Asn Val ThrGlu Asp Gln Lys Pro Leu Trp Asp Glu Ser Cys Trp 20 25 30 Ser Asp Val GluPhe Cys Arg Arg Val Lys Met Thr Gly Trp Met Tyr 35 40 45 Phe Val Ser LysThr Leu Ala Glu Gln Glu Ala Trp 50 55 60 <210> SEQ ID NO 33 <211>LENGTH: 59 <212> TYPE: PRT <213> ORGANISM: Oryza sp. <400> SEQUENCE: 33Ala Gly Thr Val Lys Arg Ile Val Phe Thr Ser Ser Ala Gly Thr Val 1 5 1015 Asn Ile Glu Glu Arg Gln Arg Pro Ser Tyr Asp His Asp Asp Trp Ser 20 2530 Asp Ile Asp Phe Cys Arg Arg Val Lys Met Thr Gly Trp Met Tyr Phe 35 4045 Val Ser Lys Ser Leu Ala Glu Lys Ala Ala Met 50 55 <210> SEQ ID NO 34<211> LENGTH: 60 <212> TYPE: PRT <213> ORGANISM: Fragaria sp. <400>SEQUENCE: 34 Lys Ala Lys Thr Val Arg Arg Leu Val Phe Thr Ser Ser Ala GlyAla 1 5 10 15 Val Ala Ile Glu Glu His Pro Lys Glu Val Tyr Ser Glu AsnAsn Trp 20 25 30 Ser Asp Val Val Phe Cys Arg Lys Val Lys Met Thr Gly TrpMet Tyr 35 40 45 Phe Val Ser Lys Thr Leu Ala Glu Gln Ala Ala Trp 50 5560 <210> SEQ ID NO 35 <211> LENGTH: 3879 <212> TYPE: DNA <213> ORGANISM:Petunia x hybrida <400> SEQUENCE: 35 attagatttt ttaatgaact ttaaacttgattccctaacc tgttgaacgt gttagggctt 60 ttgacctgaa tttttaaact attaggactcctcttattga agggatgaaa aagactccta 120 attgaaatat atctccttta tatgacttatcctttactta gaggagaagt aatagacaac 180 aataaataga tgatcttctt ctcacaatacacaacacaaa ttccacaatg tagtcttagg 240 agaattttat ttaggggaga tttttcttcccatattatgt acgcagttgg ccaaactact 300 ttcaataaca acccttttga tatgtgtcattttcatattt gattcattgt cattaatgtt 360 tgtgtgttac caaccgcatg catcatgttgttccgatccc aacaagtagt atcagagcca 420 tattcaacta atggttcgat gagccaggttataaggttga agatatgttc aaggcgggtt 480 cagagctgca accaatgacg ataataaagttatataaaaa ataggatggt aatgctacgt 540 gtggagaaaa gtttcattca accatatattcaacaataat gttgctgctg catctttaaa 600 acaaaatact ttttaaccca tgttttggctacttttaacc aatctcagtt ttaactcatg 660 cttattttaa tgcttgggct cccttttaatccattcttgg gctcattttt aacctgttgc 720 tgggcttctt tgaaccaaaa taatatttttaaacatgaca aacagcagtt tgaagaccat 780 gtgaagaagg aagatcaaga ttcttttgtccaaaattcag gccaaggcgg gaattgttag 840 tgtttttacc ctgaattttt aacctattaggactactctt attgaaggga tgaaaaagac 900 tcctaattag aatatatctc atttatatgacttatcctta gaggagaagt aatagacaac 960 aataaataga ggatcttctt ctcacaacaccaacacaaat tccacaatgt agtcttagga 1020 gaattttatt taggggagat tttttcttcccatattatgt agcccagttg gccaaactac 1080 tttcaataac aacccttttg atatgtgtcatttttatatt tgattcattg acattatgtt 1140 tgtgtgttta cgttccgcat gcaccatgttgtttcgatcc caacggaagg gacacatggt 1200 aacattcaat gccagtttct caatttcgaccaacatccaa aagtgatatt gcatatatgg 1260 atgaaaatat gtttcttcat cacggtacgactcaatgatc tttctaaaat cggaaaattt 1320 ctaaggactg catggttcga aactcaaaaatgataaatat atccctttat cattctccac 1380 taaatattag gttgttcgaa cctataaattacggctttcc acacatcacg tgttgcgtta 1440 caactaaacc aaaaccattg gaatcatgcggagccacctt tgggcaaggg aattcaattg 1500 aaccctcttc acccgaaaat ttgtactgcattgatatttt aaattttgaa cctcttattg 1560 aaaatcctgt ctccgtcctg cttggagcaacaacacaact ctatatgcat atgaaagagt 1620 gggtcctaag taaccagata ctacaccatccccacagccc cattttcttc tctctcagca 1680 accagtccta tttagttaat ccaatgaagttactcaacgg gccgttgagc acgtgctcac 1740 catctaacat tcccaatcct tagacaacctacgtgcaagt actataaaga cagatataaa 1800 ccaacacata aataaagttc atcctgttgtaatttaacta ctagtaagtc cactaaaatt 1860 aacaaaatct taagtccgac tttccaacttccatatctga taatggcaag tgaagcagtt 1920 catgcccctt cacctccggt ggcagtgccgacagtttgcg tcactggagc tgctggattt 1980 attggctctt ggcttgtcat gagactccttgaacgcggtt acaatgttca cgctactgtt 2040 cgtgatcctg gtatgttttg tttcgagagtttaacttcta tgcattgcta gcgtaaaaga 2100 actttgaaag tggtatgcgc gtgaagagaagtatgtgaca ttgataaaag tgtgcccttt 2160 gtatggcatg cacttacgta aagatgcatgattttgtaga gaacaagaag aaggtgaaac 2220 atctgctgga actgccaaag gctgatacgaacttaacact gtggaaagcg gacttgacag 2280 tagaaggaag ctttgacgag gccattcaaggctgtcaagg agtatttcat gtagcaacac 2340 ctatggattt cgagtccaaa gaccctgaggtacgatcaaa ctagaagcaa atatacttgt 2400 ggtcctttct acatttctgg tctaaattctaacataacta tgtaactacg agatatgaca 2460 gaatgaagta atcaagccaa cagtccggggaatgctaagc atcattgaat catgtgctaa 2520 agcaaacaca gtgaagaggc tggttttcacttcatctgct ggaactctcg atgtgcaaga 2580 gcaacaaaaa cttttctatg accagaccagctggagcgac ttggacttca tatatgctaa 2640 gaagatgaca ggatgggttt gtttggctattcttttcatt tcgtaataca ctctagtaac 2700 aaaaacagca ttctcattga tacttgtgaattaatttcat tgcagatgta ttttgcttcc 2760 aagatactgg cagagaaagc cgcaatggaagaagctaaaa agaagaacat tgatttcatt 2820 agcatcatac caccactggt tgttggtccattcatcacac ctacatttcc ccctagttta 2880 atcactgccc tttcactaat tactggtatgctgtagtctt aaatattcta cgtaattaaa 2940 ttgcacagat gatgtgcagt tcttcctctcaccaaacacc cacaaattat ttcaattaac 3000 aatattttta cagtcatggg tttaatcagattggggtatg cagggaatga agctcattac 3060 tgcatcatta aacaaggtca atatgtgcatttggatgatc tttgtgaggc tcacatattc 3120 ctgtatgagc accccaaggc agatggaagattcatttgct cgtcccacca tgctatcatc 3180 tacgatgtgg ctaagatggt ccgagagaaatggccagagt actatgttcc tactgagtaa 3240 gcctctctct tctgtattcc caagtatagtaggctccttc attgagtgat ggcttagtaa 3300 ctcactcgtg ggtaaataac aggtttaaagggatcgataa agacctgcca gtggtgtctt 3360 tttcatcaaa gaagctgaca gatatgggttttcagttcaa gtacactttg gaggatatgt 3420 ataaaggggc catcgatact tgtcgacagaagcagctgct tcccttttct acccgaagtg 3480 ctgaagacaa tggacataac cgagaagccattgccatttc tgctcaaaac tatgcaagtg 3540 gcaaagagaa tgcaccagtt gcaaatcatacagaaatgtt aagcaatgtt gaagtctaga 3600 actgcaatct tgacaagata aagaaagcttgccaagcaat atgtttgcta ctaagttctt 3660 tgtcatctgt ttgagggttt tcaaaactaaatcagtaaat ttttcgatgc atatagagaa 3720 gttcttgtct tgctaaatta cgggcagcctaaacaatagg atatcaagaa tcccgtgcta 3780 tatttttcag gaaaataaaa tctataatcatttcagggaa tctggatact aatacaagga 3840 cgtattttcc aatttataag ctttgcaaaagcaagatct 3879

What is claimed is:
 1. An isolated nucleic acid encoding adihydroflavanol 4-reductase that selectively catalyzes the reduction ofdihydrokaempferol to leucopelargonidin in the presence ofdihydroquercetin and dihydromyricetin comprising a nucleic acidsequence, of SEQ ID NO: 1 wherein the codon for the Asn residue atposition 134 of the encoded polypeptide has been mutated to a codon forLeu.
 2. A vector able to be introduced into a plant cell that encodes adihydroflavanol 4-reductase that selectively catalyzes the reduction ofdihydrokaempferol to leucopelargonidin in the presence ofdihydroquercetin and dihydromyricetin in a plant containing said cell,said vector comprising the nucleic acid sequence of SEQ ID NO: 1 whereinthe codon for the Asn residue at position 134 of the encoded polypeptidehas been mutated to a codon for Leu and said sequence is operably linkto a promoter.
 3. A vector according to claim 2 wherein said promoter isa promoter from cauliflower mosaic virus.